Aflatoxin G1 induced TNF-α-dependent lung inflammation to enhance DNA damage in alveolar epithelial cells

Aflatoxin G₁ (AFG₁), a member of the AF family with cytotoxic and carcinogenic properties, could cause DNA damage in alveolar type II (AT-II) cells and induce lung adenocarcinoma. Recently, we found AFG₁ could induce chronic lung inflammation associated with oxidative stress in the protumor stage. Chronic inflammation plays a critical role in cigarette smoke or benzo[a]pyrene-induced lung tissues damage. However, it is unclear whether and how AFG₁-induced lung inflammation affects DNA damage in AT-II cells. In this study, we found increased DNA damage and cytochrome P450 (CYP2A13) expression in AFG₁-induced inflamed lung tissues. Furthermore, we treated the mice with a soluble tumor necrosis factor (TNF)-α receptor and AFG₁ and found that TNF-α neutralization inhibited the AFG₁-induced chronic lung inflammation in vivo, and then reversed the CYP2A13 expression and DNA damage in AT-II cells. The results suggest that AFG₁ induces TNF-α-dependent lung inflammation to regulate 2A13 expression and enhance DNA damage in AT-II cells. Then, we treated the primary mice AT-II cells and human AT-II like cells (A549) with AFG₁ and TNF-α and found that TNF-α enhanced the AFG₁-induced DNA damage in mice AT-II cells as well as A549 cells in vitro. In AFG₁-exposed A549 cells, TNF-α-enhanced DNA damage and apoptosis were reversed by CYP2A13 small interfering RNA. Blocking NF-κB pathway inhibited the TNF-α-enhanced CYP2A13 upregulation and DNA damage confirming that the CYP2A13 upregulation by TNF-α plays an essential role in the activation of AFG₁ under inflammatory conditions. Taken together, our findings suggest that AFG₁ induces TNF-α-dependent lung inflammation, which upregulates CYP2A13 to promote the metabolic activation of AFG₁ and enhance oxidative DNA damage in AT-II cells.     

Claudin 1 mediates TNFα-induced gene expression and cell migration in human lung carcinoma cells

Epithelial-mesenchymal transition (EMT) is an important mechanism in carcinogenesis. To determine the mechanisms that are involved in the regulation of EMT, it is crucial to develop new biomarkers and therapeutic targets towards cancers. In this study, when TGFβ1 and TNFα were used to induce EMT in human lung carcinoma A549 cells, we found an increase in an epithelial cell tight junction marker, Claudin 1. We further identified that it was the TNFα and not the TGFβ1 that induced the fibroblast-like morphology changes. TNFα also caused the increase in Claudin-1 gene expression and protein levels in Triton X-100 soluble cytoplasm fraction. Down-regulation of Claudin-1, using small interfering RNA (siRNA), inhibited 75% of TNFα-induced gene expression changes. Claudin-1 siRNA effectively blocked TNFα-induced molecular functional networks related to inflammation and cell movement. Claudin-1 siRNA was able to significantly reduce TNF-enhanced cell migration and fibroblast-like morphology. Furthermore, over expression of Claudin 1 with a Claudin 1-pcDNA3.1/V5-His vector enhanced cell migration. In conclusion, these observations indicate that Claudin 1 acts as a critical signal mediator in TNFα-induced gene expression and cell migration in human lung cancer cells. Further analyses of these cellular processes may be helpful in developing novel therapeutic strategies.     

Mitogenic response of rat lung and tracheal epithelial cells in monolayer primary cultures—Modulation of TNF-α expression

Epithelial cells isolated from rat lung and trachea were grown on monolayers and their response to a number of hormones and growth factors were studied. Maximum proliferative response in serum containing media was observed when insulin, cholera toxin and cortisol were present together. However, these additives when present independently showed a marginal response. The synergism, due to these factors in promoting growth was seen very early in culture (day 4) as shown by thymidine labelling studies, On examining the indices of early mitogenesis, such as the expression ofc-myc, our data suggests that these factors stimulate the expression ofc-myc within 4 h. With respect to expression of TNF-α mRNA, this study suggests a possible modulation of TNF-α expression in response to these mitogens that stimulate proliferation maximally. Whether this expression of TNF-α by these epithelial cells is due to a maximal proliferative stimulus and/or is an early step in the cascade of intracellular signalling events is to be investigated in detail.     

Cl-IB-MECA enhances TNF-α release in peritoneal macrophages stimulated with LPS

Adenosine receptor A3 (A3R) belongs to the Gi/Gq-coupled receptor family, that leads to the intracellular cAMP reduction and intracellular calcium increase, respectively. A3R is widely expressed and it can play a crucial role in many patho-physiological conditions, including inflammation. Here we investigate the effect of Cl-IB-MECA, A3R agonist, on the production of TNF-α. We found that Cl-IB-MECA enhances LPS-induced TNF-α release in peritoneal macrophages. This effect is reduced by MRS1191, A3R antagonist and by forskolin, activator of adenylyl cyclase. pIκBα increased in LPS+Cl-IB-MECA-treated macrophages, while total IκB kinase-β (IKKβ) reduced. Indeed, p65NF-κB nuclear translocation increased in cells treated with LPS+Cl-IB-MECA. Moreover, IMD 0354, IKKβ inhibitor, significantly abrogated the effect of Cl-IB-MECA on TNF-α release. Inhibition of protein kinase C (PKC) significantly reduced Cl-IB-MECA-induced TNF-α release in LPS-stimulated macrophages. Furthermore, LY-294002, PI3K inhibitor, reduced the TNF-α production enhanced by Cl-IB-MECA, although the phosphorylation status of Akt did not change in cells treated with LPS+Cl-IB-MECA than LPS alone. In summary, these data show that Cl-IB-MECA is able to enhance TNF-α production in LPS-treated macrophages in an NF-κB- dependent manner.     

Lactobacillus plantarum lipoteichoic acid alleviates TNF-α-induced inflammation in the HT-29 intestinal epithelial cell line

We recently observed that lipoteichoic acid (LTA) isolated from Lactobacillus plantarum inhibited endotoxin-mediated inflammation of the immune cells and septic shock in a mouse model. Here, we examined the inhibitory role of L. plantarum LTA (pLTA) on the inflammatory responses of intestinal epithelial cells (IEC). The human colon cell line, HT-29, increased interleukin (IL)-8 expression in response to recombinant human tumor necrosis factor (TNF)-alpha, but not in response to bacterial ligands and interferon (IFN)-gamma. TNF-α also increased the production of inducible nitric oxide synthase (iNOS), nitric oxide (NO), and intercellular adhesion molecule 1 (ICAM-1) through activation of p38 mitogen-activated protein kinase (MAPK) from HT-29 cells. However, the inflammatory response of HT-29 on TNF-α stimulation was significantly inhibited by pLTA treatment. This pLTA-mediated inhibition accompanied the inhibition of nuclear factor (NF)-kappa B and MAPKs. Our data suggest that pLTA regulates cytokine-mediated immune responses and may be a good candidate for maintaining intestinal homeostasis against excessive inflammation.     

Scribble is required for normal epithelial cell–cell contacts and lumen morphogenesis in the mammalian lung

During lung development, proper epithelial cell arrangements are critical for the formation of an arborized network of tubes. Each tube requires a lumen, the diameter of which must be tightly regulated to enable optimal lung function. Lung branching and lumen morphogenesis require close epithelial cell–cell contacts that are maintained as a result of adherens junctions, tight junctions and by intact apical–basal (A/B) polarity. However, the molecular mechanisms that maintain epithelial cohesion and lumen diameter in the mammalian lung are unknown. Here we show that Scribble, a protein implicated in planar cell polarity (PCP) signalling, is necessary for normal lung morphogenesis. Lungs of the Scrib mouse mutant Circletail (Crc) are abnormally shaped with fewer airways, and these airways often lack a visible, ‘open’ lumen. Mechanistically we show that Scrib genetically interacts with the core PCP gene Vangl2 in the developing lung and that the distribution of PCP pathway proteins and Rho mediated cytoskeletal modification is perturbed in Scrib^Crc/Crc lungs. However A/B polarity, which is disrupted in Drosophila Scrib mutants, is largely unaffected. Notably, we find that Scrib mediates functions not attributed to other PCP proteins in the lung. Specifically, Scrib localises to both adherens and tight junctions of lung epithelia and knockdown of Scrib in lung explants and organotypic cultures leads to reduced cohesion of lung epithelial cells. Live imaging of Scrib knockdown lungs shows that Scrib does not affect bud bifurcation, as previously shown for the PCP protein Celsr1, but is required to maintain epithelial cohesion. To understand the mechanism leading to reduced cell–cell association, we show that Scrib associates with β-catenin in embryonic lung and the sub-cellular distribution of adherens and tight junction proteins is perturbed in mutant lung epithelia. Our data reveal that Scrib is required for normal lung epithelial organisation and lumen morphogenesis by maintaining cell–cell contacts. Thus we reveal novel and important roles for Scrib in lung development operating via the PCP pathway, and in regulating junctional complexes and cell cohesion.     

Chidamide alleviates TGF-β-induced epithelial–mesenchymal transition in lung cancer cell lines

Transforming growth factor-β (TGF-β)-induced epithelial–mesenchymal transition is a critical process in the initiation of metastasis of various types of cancer. Chidamide is a class I histone deacetylase inhibitor with anti-tumor activity. This study investigated the effects of chidamide on TGF-β-mediated suppression of E-cadherin expression in adenocarcinomic lung epithelial cells and the molecular mechanisms involved in these effects. Western blot analysis, confocal microscopy, Quantitative methyl-specific PCR and bisulfite sequencing were used to evaluate the effects of different treatments on chidamide ameliorating TGF-β induced-E-cadherin loss. H3 acetylation binding to the promoter of E-cadherin was detected by chromatin immunoprecipitations (CHIP). We found that chidamide reduced the level of lung cancer cell migration observed using a Boyden chamber assay (as an indicator of metastatic potential). Chidamide inhibited TGF-β-induced SMAD2 phosphorylation and attenuated TGF-β-induced loss of E-cadherin expression in lung cancer cells by Western blotting and confocal microscopy, respectively. Quantitative methyl-specific PCR and bisulfite sequencing revealed that TGF-β-enhanced E-cadherin promoter methylation was ameliorated in cells treated with chidamide. We demonstrated that histone H3 deacetylation within the E-cadherin promoter was required for TGF-β-induced E-cadherin loss; cell treatment with chidamide increased the H3 acetylation detected by CHIP. Taken together, our results demonstrate that TGF-β suppressed E-cadherin expression by regulating promoter methylation and histone H3 acetylation. Chidamide significantly enhanced E-cadherin expression in TGF-β-treated cells and inhibited lung cancer cell migration. These findings indicate that chidamide has a potential therapeutic use due to its capacity to prevent cancer cell metastasis.     

The role of IFN-γ in regulation of IFN-γ-inducible protein 10 (IP-10) expression in lung epithelial cell and peripheral blood mononuclear cell co-cultures

Background

Epithelial to mesenchymal transition (EMT) in alveolar epithelial cells (AECs) has been widely observed in patients suffering interstitial pulmonary fibrosis. In vitro studies have also demonstrated that AECs could convert into myofibroblasts following exposure to TGF-β1. In this study, we examined whether EMT occurs in bleomycin (BLM) induced pulmonary fibrosis, and the involvement of bronchial epithelial cells (BECs) in the EMT. Using an α-smooth muscle actin-Cre transgenic mouse (α-SMA-Cre/R26R) strain, we labelled myofibroblasts in vivo. We also performed a phenotypic analysis of human BEC lines during TGF-β1 stimulation in vitro.

Methods

We generated the α-SMA-Cre mouse strain by pronuclear microinjection with a Cre recombinase cDNA driven by the mouse α-smooth muscle actin (α-SMA) promoter. α-SMA-Cre mice were crossed with the Cre-dependent LacZ expressing strain R26R to produce the double transgenic strain α-SMA-Cre/R26R. β-galactosidase (βgal) staining, α-SMA and smooth muscle myosin heavy chains immunostaining were carried out simultaneously to confirm the specificity of expression of the transgenic reporter within smooth muscle cells (SMCs) under physiological conditions. BLM-induced peribronchial fibrosis in α-SMA-Cre/R26R mice was examined by pulmonary βgal staining and α-SMA immunofluorescence staining. To confirm in vivo observations of BECs undergoing EMT, we stimulated human BEC line 16HBE with TGF-β1 and examined the localization of the myofibroblast markers α-SMA and F-actin, and the epithelial marker E-cadherin by immunofluorescence.

Results

βgal staining in organs of healthy α-SMA-Cre/R26R mice corresponded with the distribution of SMCs, as confirmed by α-SMA and SM-MHC immunostaining. BLM-treated mice showed significantly enhanced βgal staining in subepithelial areas in bronchi, terminal bronchioles and walls of pulmonary vessels. Some AECs in certain peribronchial areas or even a small subset of BECs were also positively stained, as confirmed by α-SMA immunostaining. In vitro, addition of TGF-β1 to 16HBE cells could also stimulate the expression of α-SMA and F-actin, while E-cadherin was decreased, consistent with an EMT.

Conclusion

We observed airway EMT in BLM-induced peribronchial fibrosis mice. BECs, like AECs, have the capacity to undergo EMT and to contribute to mesenchymal expansion in pulmonary fibrosis.     

Immuno-localization of type-IV collagen in the blood-gas barrier and the epithelial–epithelial cell connections of the avian lung

The terminal respiratory units of the gas exchange tissue of the avian lung, the air capillaries (ACs) and the blood capillaries (BCs), are small and rigid: the basis of this mechanical feature has been highly contentious. Because the strength of the blood-gas barrier (BGB) of the mammalian lung has been attributed to the presence of type-IV collagen (T-IVc), localization of T-IVc in the basement membranes (BM) of the BGB and the epithelial–epithelial cell connections (E-ECCs) of the exchange tissue of the lung of the avian (chicken) lung was performed in order to determine whether it may likewise contribute to the strength of the BGB. T-IVc was localized in both the BM and the E-ECCs. As part of an integrated fibroskeletal scaffold on the lung, T-IVc may directly contribute to the strengths of the ACs and the BCs.     

Resistin up-regulates fractalkine expression in human endothelial cells: Lack of additive effect with TNF-α

Resistin is a cytokine and fractalkine (Fk) a cell adhesion molecule and chemokine that contribute to human vascular inflammation by mechanisms not clearly defined. We questioned whether resistin induces Fk expression in human endothelial cells (HEC), compared the effect with that of the pro-inflammatory cytokine, TNF-α, and evaluated the consequences of co-stimulating HEC with both activators on Fk induction and on the signalling molecules involved. We found that resistin up-regulated Fk expression at comparable level to that of TNF-α by a mechanism involving P38 and JNK MAPK and NF-κB. Co-stimulation of cells with resistin and TNF-α did not increase Fk expression induced by every single inducer. Moreover resistin reduced the expression induced by TNF-α in HEC. The new data uncover Fk as a novel molecular link between resistin and inflammation and show that resistin and TNF-α have no additive effect in Fk up-regulation or on the signalling molecules implicated.     

Elevated ZIPK is required for TNF-α-induced cell adhesion molecule expression and leucocyte adhesion in endothelial cells

Leucocyte adhesion to the vascular endothelium is a critical event in the early inflammatory response to infection and injury.This process is primarily regulate...     

Is downregulation of MHC class I antigen expression in human non-small cell lung cancer associated with prolonged survival?

Purpose: To characterize HLA class I antigen expression in non-small cell lung cancer (NSCLC) lesions, and to assess the clinical significance of these molecules’ downregulation. Methods: One hundred and ninety primary formalin fixed, paraffin embedded NSCLC lesions were stained with HLA class I heavy chain-specific mAb HC-10. Results were scored as percentage of stained tumor cells and categorized into three groups: 0–24% (negative), 25–75% (heterogeneous) and >75% (positive). HLA class I antigen expression was correlated with clinical and pathologic predictors of time to progression and survival and analyzed using the chi-square test. Association between HLA class I antigen expression and survival was assessed using Cox regression models, while controlling for confounders. Results: HLA class I antigen expression was negative, heterogeneous and positive in 153, 25 and 12 primary NSCLC lesions, respectively. Independent variables significantly associated with survival included tumor stage, PS and weight loss. The median survival times were 40.6, 44.0 and 17.9 months for patients with a HLA class I antigen expression scored as negative, heterogeneous and positive, respectively. Conclusion: HLA class I antigen defects were found with high frequency (93.6%) in NSCLC lesions. HLA class I antigen downregulation was associated with improved survival, although this association was not statistically significant. These results, which parallel similar findings in uveal melanoma and in breast carcinoma, raise the possibility that NK cells may play a role in the control of NSCLC tumors.N. Ramnath and D. Tan contributed equally to the paper     

Ectopic expression of Ligand-of-Numb protein X promoted TGF-β induced epithelial to mesenchymal transition of proximal tubular epithelial cells

Ligand-of-Numb protein X (LNX) was initially characterized as a RING finger type E3 ubiquitin ligase that targeted the intrinsic cell fate determinant Numb for ubiquitination dependent degradation. However, the physiological function of LNX remains largely unknown. In the present study, we demonstrate that ectopic expression of LNX in human proximal tubular epithelial cells (HK-2 cells) significantly enhanced TGF-β1 induced epithelial to mesenchymal transition (EMT). The EMT-promoting effect of LNX manifested as strong inhibition of E-cadherin expression, enhanced expression of vimentin, fibronectin or PAI-1, and increased cell migration. This function of LNX was shown to be independent of its ligase activity because ectopic expression of a mutant form of LNX (C48ALNX) that lacks E3 ligase activity had the similar effect as the wild-type LNX. Overexpression of E-cadherin could inhibit LNX augmented EMT. This study suggests a potential role for LNX in promoting EMT in human proximal tubular epithelial cells.